Stable liquid interferon beta formulations

ABSTRACT

Liquid interferon compositions having a pH between 4.0 and 7.2 are described. The compositions comprise interferon-beta and a stabilizing agent at between about 0.3% and 5% by weight which is an amino acid selected from the group consisting of acidic amino acids, arginine and glycine. If needed, salt is added to provide sufficient ionic strength. The liquid composition has not been previously lyophilized or previously cavitated. The liquid is preferably contained within a vessel having at least one surface in contract with the liquid that is coated with a material inert to adsorption of interferon-beta. A kit for parenteral administration of a liquid interferon formulation and a method for stabilizing liquid interferon compositions are also described.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/794,477, filed Jun. 4, 2010, which is a continuation of U.S. patentapplication Ser. No. 11/975,379, filed Oct. 18, 2007, which is acontinuation of U.S. patent application Ser. No. 10/397,108, filed Mar.24, 2003 (now abandoned), which is a continuation of U.S. patentapplication Ser. No. 09/403,930, filed May 19, 2000 (now abandoned),which is a National Phase filing under 35 U.S.C. §371 of InternationalApplication PCT/US97/23817, filed Dec. 23, 1997, which designates theUnited States, is published in English and claims priority under 35U.S.C. §119(e) from U.S. Provisional Application 60/034,353, filed Dec.24, 1996. The specifications of each of the foregoing applications areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to methods for stabilizing human interferon-betaand to stable, interferon-beta liquid formulations.

BACKGROUND OF THE INVENTION

Interferons are proteins having a variety of biological activities, someof which are antiviral, immunomodulating and antiproliferative. They arerelatively small, species-specific, single chain polypeptides, producedby mammalian cells in response to exposure to a variety of inducers suchas viruses, polypeptides, mitogens and the like. Interferons protectanimal tissues and cells against viral attack and are an important hostdefense mechanism. In most cases, interferons provide better protectionto tissues and cells of the kind from which they have been produced thanto other types of tissues and cells, indicating that human-derivedinterferon should be more efficacious in treating human diseases thaninterferons from other species.

There are several distinct types of human interferons, generallyclassified as leukocyte (interferon-alpha), fibroblast (interferon-beta)and immune (interferon-gamma), and a large number of variants thereof.General discussions of interferons can be found in various texts andmonographs including: The Interferon System (W. E. Stewart, II,Springer-Verlag, N.Y. 1979); and Interferon Therapy (World HealthOrganization Technical Reports Series 676, World Health Organization,Geneva 1982), incorporated herein by reference.

The method of administering interferon is an important factor in theclinical application of this important therapeutic agent. Systemicadministration of interferon by either intravenous, intramuscular orsubcutaneous injection has been most frequently used with some successin treating disorders such as hairy cell leukemia, Acquired ImmuneDeficiency Syndrome (AIDS) and related Kaposi's sarcoma. It is known,however, that proteins in their purified form are especially susceptibleto degradation. For interferon-beta, the primary mechanism(s) ofinterferon degradation in solution are aggregation and deamidation. Thelack of interferon stability in solutions and other products hasheretofore limited its utility.

Pharmaceutical interferon compositions for clinical use commonly containinterferon as a lyophilized (i.e., freeze-dried) preparation incombination with complex organic excipients and stabilizers such asnonionic surface active agents (i.e., surfactants), various sugars,organic polyols and/or human serum albumin. Lyophilized preparationshave the disadvantage of requiring complex packaging since a separatesupply of sterile water for injection is required. Moreover, lyophilizedpreparations require several manipulations prior to use, thus increasingthe possibility of needle sticks and dropped components duringpreparation for injection. These manipulations are especiallyproblematic for patient populations that exhibit muscle weakness andpoor coordination, such as people with multiple sclerosis (MS). MSpatients may self-administer interferons so that the availability of adosage form that is much easier to administer than current lyophilizedproducts represents important added value for the target patientpopulation. Simple liquid formulations of interferon are highlydesirable in order to avoid the reconstitution necessary whenlyophilized preparations are used.

Liquid, non-lyophilized formulations containing interferons may alsocontain complex carriers such as human serum albumin, polyols, sugars,and anionic surface active stabilizing agents. See, for example, WO89/10756 (Hara et al.—containing polyol and p-hydroxybenzoate).

SUMMARY OF THE INVENTION

This invention has solved the above problems with the discovery thathuman interferon-beta can be stabilized when placed in bufferedsolutions having a pH between about 4 and 7.2, the solutions containingan amino acid as a stabilizing agent and in some cases a salt (if theamino acid does not contain a charged side chain). The interferon-betais not lyophilized but, once prepared from sources using methods knownto the ordinarily skilled artisan, is included directly in theformulation of this invention.

Therefore, one aspect of the invention is a liquid compositioncomprising an interferon and a stabilizing agent at between about 0.3%and 5% by weight which is an amino acid selected from the groupconsisting of acidic amino acids, arginine and glycine. The liquidcomposition has not been previously lyophilized. Moreover, it ispreferable that the liquid composition is contained within a vessel,such as a syringe, in which the vessel has a surface in contact with theliquid that is coated with a material inert to interferon such assilicone or polytetrafluoroethylene. Preferred compositions includeinterferon-beta, or a recombinantly produced interferon, in a bufferhaving a pH between about 4.0 and about 7.2. Other formulations of theinvention include:

(1) a 20 mM acetate buffer at pH 5.0, the buffer not previouslylyophilized, in which the buffer includes interferon-beta plusingredients selected from (a) 150 mM arginine-HC1; (b) 100 mM sodiumchloride and 70 mM glycine; (c) 150 mM arginine-HCl and 15 mg/ml humanserum albumin; (d) 150 mM arginine-HCl and 0.1% PLURONIC® F-68surfactant; (e) 140 mM sodium chloride; (1) 140 mM sodium chloride and15 mg/ml human serum albumin; and (g) 140 mM sodium chloride and 0.1%PLURONIC® F-68 surfactant;

(2) a liquid at pH 5.0 that includes interferon-beta, 170 mM L-glutamicacid, and 150 mM sodium hydroxide, the liquid not previouslylyophilized;

(3) a 20 mM phosphate buffer at pH 7.2, the buffer not previouslylyophilized, wherein the buffer includes interferon-beta plusingredients selected from: (a) 140 mM arginine-HCl; and (b) 100 mMsodium chloride and 70 mM glycine.

Another embodiment of the invention is a kit for parenteraladministration of a liquid interferon formulation. The kit comprises avessel containing a liquid formulation at a pH of between 4 and 6, theliquid comprising a pharmaceutically effective amount of interferon-betathat has not been previously lyophilized and an amino acid stabilizingagent about 5% by weight or less; and instructions for use.

Yet another embodiment of the invention is a liquid pharmaceuticalcomposition suitable for parenteral administration to mammals consistingessentially of an effective amount of interferon-beta that has not beenpreviously lyophilized in a buffer maintaining the pH within the rangeof 4.0 to 6.0, and an amino acid stabilizing agent at an appropriateionic strength. The composition is contained within a storage vesselsuch as a syringe. Preferably, the storage vessel lacks anoxygen-containing/liquid interface (i.e, the interferon solution is notsubjected to oxygen containing gas during preparation and storage). Theinterferon-beta essentially retains its antiviral activity duringstorage at a temperature of between about 2 degrees C. and about 25degrees C. for a period of at least 3 months.

A process of the invention for stabilizing interferon-beta in liquidpharmaceutical compositions so that it essentially retains its physicalstability during storage at a temperature of between about 2 and about25 degrees C. for a period of at least 3 months, comprises admixing: a)an effective amount of interferon-beta; b) a buffer maintaining the pHwithin the range of 4.0 to 7.2 at an appropriate ionic strength; and c)an amino acid stabilizing agent, wherein the liquid has previously notbeen lyophilized and has not been subject to oxygen containing gasduring preparation and storage.

The liquid formulations of the invention have many advantages overlyophilized formulations. The advantages include: (i) a smallerinjection volume required for a liquid formulation will induce lessdiscomfort than a larger volume; (ii) replacement of complex excipientswith simple amino acids makes it possible to monitor finished productquality more closely; (iii) packaging is greatly simplified due toelimination of the need for a separate supply of water for injection(WFI) and separate syringe and vial; (iv) dosing accuracy may beimproved due to fewer liquid transfers; and (v) product safety isimproved because the simpler administration decreases the chance ofneedle punctures and dropped components during preparation forinjection.

Therefore, an object of the present invention is to provide abiologically active, stable liquid formulation of interferon-beta foruse in injectable applications.

Another object of this invention is to provide a formulation which doesnot require prior lyophilization of a interferon-beta composition.

It is another object of this invention to prevent loss of stability inan interferon-beta liquid formulation by: a) avoiding cavitation and/orhead space formation during preparation of the liquid composition, or b)storing the liquid formulation with a head space that consists of aninert gas such as argon or nitrogen.

Still another object of this invention is to provide a liquidformulation permitting storage for a long period of time in a liquidstate facilitating storage and shipping prior to administration. Anotherobject of this invention is to provide a liquid formulation which iseasily made and administered having eliminated lyophilization andreconstitution steps.

A further object of the invention is the use of simple amino acids asalternate stabilizers besides commonly-used serum albumin, making iteasier to monitor product quality.

Yet another object of this invention is to provide a pharmaceuticalcomposition containing non-lyophilized interferon-beta that can beproduced less expensively.

Other advantages of the invention are set forth in part in thedescription which follows, and in part, will be obvious from thisdescription, or may be learned from the practice of this invention. Theaccompanying drawings, which are incorporated in, and constitute a partof, this specification, illustrates and together with this description,serves to explain the principle of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the percentage of interferon-beta monomerremaining in bulk process liquid as a function of the percentagedissolved oxygen in the liquid.

FIG. 2 is a graph showing the percentage of protein concentrationnormalized against that of starting material versus time for liquidformulation BG9589-1. Samples labeled “4° C.” (closed squares) areincubated at between 2-8° C. Other samples are incubated at 25° C.(closed circles); 33° C. (closed triangles) and 40° C. (closed diamonds)

FIG. 3 is a graph showing the percentage of protein concentrationnormalized against that of starting material versus time for liquidformulation BG9589-3. Samples labeled “4° C.” are incubated at between2-8° C. Other samples are incubated at 25° C. (closed circles); 33° C.(closed triangles) and 40° C. (closed diamonds).

DETAILED DESCRIPTION OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides a simplemethod for stabilizing interferon and a simple interferon formulationwith enhanced storage stability. The invention is based, in part, on ourdiscoveries that:

a) interferon-beta is particularly unstable and aggregates whencontacted with oxygen that is either actively bubbled through the liquidor statically contacted as in a head space;

b) interferon-beta liquid preparations lacking a carrier such as humanserum albumin are particularly susceptible to adsorption (i.e., eitherchemical reaction or physical linkage) to the glass surfaces; and

c) interferon-beta aggregates at low ionic strength, requiring an ionicmilieu for stability in the aqueous state.

The invention is therefore directed to methods for stabilizing humaninterferon-beta that avoid these pitfalls, and to the resulting liquidformulations of stabilized interferon-beta.

A. Definitions

The term “buffer” refers to solutions of a weak acid and a saltcontaining the anion of the acid, or solutions of a weak base and itssalt. In particular, the term “acetate” when used in this specification(see also Table I, infra) refers to a buffer system preferablycontaining sodium acetate and acetic acid and the term “phosphate”refers to a buffer system preferably containing dibasic and monobasicsodium phosphate hepta- and mono-hydrate, respectively. Moreover, thosesolutions in Table II (infra) containing an acidic amino acid incombination with sodium hydroxide, albeit not conventionally consideredto be buffers as this term is known in the art, are nonetheless includedwithin the definition herein.

The term “excipient” refers to any compound added during processingand/or storage to a liquid formulation for the purpose of altering thebulk properties, improving stability and/or adjustment of osmolality.

The term “stabilizing agent” refers to an excipient that improves orotherwise enhances stability.

The term “stability” has by necessity a functional definition and meansthe relative temporal constancy of interferon activity such asanti-viral activity and/or interferon structure.

The term “cavitated” refers to any liquid interferon formulation that,because of changes in pressure or physical agitation, has had contactwith oxygen-containing bubbles (e.g., air) at least during itspreparation and storage. The term “cavitation” also means that aoxygen-containing gas/liquid interface has been formed at some pointduring preparation, storage, and use of the liquid interferonformulation. The term “cavitated” also means that the dissolved oxygenlevels in the liquid interferon formulations exceed about 10% ofatmospheric equilibrium values at the temperatures typically encounteredat least during preparation and storage.

The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intrasternal, intraperitoneal, ophthalmic, or intraspinalinjection or infusion techniques.

The expression “pharmaceutically-acceptable salt” means any organic orinorganic addition salt which is relatively non-toxic and innocuous to apatient at concentrations consistent with effective activity so that theside effects ascribable to the salt do not vitiate the beneficialeffects of the interferon.

An “effective amount” of a compound is that amount which produces aresult or exerts an influence on the particular condition being treated.An “effective amount” also means that amount which produces a positiveresult (i.e., exerts an anti-viral effect) in the CPE test foranti-viral activity.

As used herein, a “pharmaceutically effective amount” of interferonmeans a percentage concentration of that agent known in the medical andpharmaceutical arts to be safe and effective in treating a particularcondition.

“Isotonic to blood” (used interchangeably with “isotonicity”) refers toa liquid interferon composition that has a sufficient concentration ofcomponents so that its osmotic behavior is substantially identical toblood, i.e., cells in contact with the formulation will substantiallyretain their shape and undergo substantially no net transfer of water byosmotic pressures.

“Poly-ionic species” (used interchangeable with “polyelectrolyticspecies”) refers to a substance of high molecular weight that is anelectrolyte and, when used in the formulations of this invention,maximizes ionic strength for a given osmolality. This definition isbased on our finding that interferon-beta is stabilized by high ionicstrength, but that total ionic strength is limited by the necessity forthe solution to be isotonic to blood (See Example 7). A preferred way tomaximize ionic strength for a given osmolality is to use an excipientthat is a poly-ionic species.

A material that is “inert to interferon” means a material having atleast the property of not physically and/or chemically reacting withinterferon.

B. Making Interferons

This invention is generally applicable to all types of interferonincluding natural interferon, interferon produced by recombinant DNAtechnology, and interferon produced by chemical synthesis ormodification. Also, the invention can be used with crude, semi-purifiedand purified interferon from fibroblasts, leukocytes, lymphocytes or anyother interferon-containing or producing tissues from humans or anyother appropriate species. Most preferably, the invention is applicableto human fibroblast interferon (interferon-beta).

The most preferred interferon-beta is a recombinant form and recombinantDNA methods for producing proteins including the various interferons areknown and are not intended to limit the invention in any way. See forexample, U.S. Pat. Nos. 4,399,216, 5,149,636, 5,179,017 (Axel et al);4,470,461 (Kaufman). Recombinant forms of interferon-beta have beenproduced. See, for example, European Patent 0 41313 (Fiers—expression ofinterferon-beta); U.S. Pat. No. 4,966,843 (McMormick et al.—expressionof interferon in CHO cells); U.S. Pat. No. 5,326,859 (Sugano et al.—DNAencoding interferon-beta); Interferon-beta can also be modified, eitherrecombinantly or chemically and can be produced in serum-containing orserum-free media. Forms of interferon-beta may include variants such ascysteine-depleted mutants (U.S. Pat. Nos. 4,588,585 and 4,737,462:Market al.) and methionine-depleted mutants (EP 260 350—Wang et al.). Theprimary amino acid sequence of the protein may be augmented byderivatization using sugar moieties (glycosylation) or by othersupplementary molecules. Other modifications may take place through thepost-translational processing systems of the host cell. Individual aminoacid residues in the chain may be further modified by oxidation,reduction or other derivatization, and the protein may be cleaved toobtain active fragments. The exact chemical structure of a particularrecombinant interferon-beta will therefore depend a several factors andis not intended to limit the scope of the invention. All suchinterferon-beta proteins included in the formulations described hereinwill retain their bioactivity when placed in suitable environmentalconditions.

One method of producing recombinant interferon-beta is to cultureChinese hamster ovary (CHO) cells transfected with the humaninterferon-beta gene. Recombinant interferon-beta is secreted by CHOcells grown up in batch suspension culture containing fetal bovineserum. Cells may be grown in spinner flasks housed in a CO₂ incubator(5% CO₂) at about 35 degrees Celsius (hereinafter “C”). Multiple spinnerflasks may be pooled and inoculated into fermenters of increasing sizeif scale-up is desired. Growth in a given fermenter is carried out forabout six days at which time the active interferon-beta productaccumulates in the culture medium. The culture may then be harvested andthe cells removed from the product-containing medium by, for example,tangential flow filtration.

C. Purifying Interferons

Purification schemes for interferons are well characterized andavailable to those having ordinary skill in the art. Such methodsinclude single- or multi-step procedures involving variouschromatographic separation steps. See, for example, U.S. Pat. No.5,015,730 (Friesen et al.—affinity chromatography and HPLC); U.S. Pat.No. 4,541,952 (Hosoi et al.—chelation chromatography).

An exemplary method involves exploiting the unusually hydrophobic andrelatively basic nature of the interferon-beta molecule as well as itsstrong affinity for binding metal ions. See, for example, Knight andFahey, “Human Fibroblast Interferon, an Improved Purification”, J. Biol.Chem., 256: 3609-3611 (1981) and Edy et al., “Purification of HumanFibroblast Interferon by Zinc Chelate Chromatography”, J. Biol. Chem.,232: 5934-5935 (1981), both of which are incorporated herein byreference.

Briefly, the capture and purification steps involve binding ofinterferon-beta to a series of SEPHAROSE® agarose bead columns (mfg. byPharmacia Biotech) and elution with salts and a polyol. Once the finalSEPHAROSE® agarose bead eluate has been diluted and adjusted by loweringpH, the interferon-beta therein will bind to SP SEPHAROSE® agarose bead(Pharmacia Biotech). Most of the remaining proteins present in thecolumn load are more basic in nature than monomeric interferon-beta andbind more tightly to the column than does the interferon. DNA andviruses partition from interferon-beta on this column. The column isthen washed with a series of buffers containing sodium chloride.

The interferon product will now bind to a chelating SEPHAROSE agarosebead (Pharmacia Biotech) column that has been previously charged withzinc. See Edy et al., supra. This column is operated under anoxygen-free atmosphere to protect the free sulfhydryl group in themolecule, as are all the subsequent steps. The purified interferon isacidified and held at low pH to inactivate any remaining viruses. Afterneutralization, the interferon is concentrated using cross flowfiltration and then buffer exchanged into a neutral buffer solution. Thebuffer exchange process reduces the concentrations of zinc and organiccompounds. Following this, the bulk interferon may be stored at −70 Cprior to the formulation steps.

D. Formulating Interferons

In the exemplary purification method described above and after the firstbuffer exchange process, a second buffer exchange process is initiatedexcept that the neutral buffer solution is replaced with a buffersolution between pH 4 and 7.2 that contains a stabilizing agent,described in more detail below. The resulting formulation containinginterferon is referred to as a “process intermediate” and may be frozenfor storage. See also Example 7.

If stored in a frozen state (under an atmosphere of an inert gas such asargon or nitrogen), it may then be thawed and pumped through a 0.22micron filter into a tared vessel, preferably stainless steel, where theprocess intermediate is combined with a previously filter-sterilizeddiluent until the desired final product weight is achieved. The diluentconsists of the same buffer that was used in the second buffer exchangeprocess. The liquid final product is then filter sterilized underaseptic procedures, using for example, two 0.22 micron filters inseries, and dispensed into a sealed vessel, preferably stainless steel,that contains an inert gas inlet, a de-gassing valve/filter combination,and an inflow/outflow dip tube. The final product is pumped through thedip tube and into the sealed vessel. Using an inert gas such asnitrogen, the final product is pressure transferred to the pump head ofa device capable of aseptically filling sterile syringes.

Several methods of aseptically filling sterile syringes are availableand the particular method used is not intended to limit the scope of thepresent invention. An exemplary method involves use of a HYPAK®autoclavable syringe filler (Becton Dickinson Pharmaceutical Systems,Franklin Lakes, N.J.). The syringes are autoclaved with tip caps inplace. Generally, devices of this type incorporate a vacuum chamber thatcontains the syringes to be filled with interferon formulation. Thechamber is placed in an aseptic environment. Each syringe liesvertically in the chamber with its open end being mated to a plungerpin, adapted to fit into the open end of the syringe barrel. The pin isdesigned to insert a stopper into the barrel to trap the liquid within.A small head space is left in the syringe after insertion. The chamberis evacuated and back-flushed with an inert, oxygen-free gas (e.g.,argon, nitrogen) several times and when the final vacuum is reached, thepins are mechanically driven into the open syringe barrels a shortdistance and the stoppers are automatically inserted into the respectivesyringes. The chamber is then vented with filtered air to bring thepressure inside the chamber back to atmospheric levels. The amount ofthe vacuum will determine the size of the inert gas-containing headspace.

In the particular system we use, the syringes are oriented verticallyand held in place by a sprocket on a rotating disk. The syringes arefirst positioned under a needle which is inserted into the syringe. Theneedle flushes the syringe interior with an inert gas (e.g. nitrogen,argon). The needle then retracts out of the syringe. The syringe is thenpositioned under a second needle which is inserted into the syringe.This needle is attached to a pump which dispenses product into thesyringe. The second needle then retracts out of the syringe. The syringeis then positioned under a third needle which is inserted into thesyringe. A plunger (previously autoclaved) is blown into the syringewith an inert, oxygen free gas (e.g. nitrogen, argon), then the needleretracts out of the syringe. The plunger is positioned to leave a headspace of inert gas between the top of the liquid and the bottom of theplunger.

1. The Excipient

The excipient is preferably a poly-ionic species that maximizes ionicstrength for a given osmolality, such as, for example, a polyelectrolytethat may include heparin or other polymeric species. As discussed inExample 4, interferon-beta is stabilized by high ionic strength, buttotal ionic strength is limited by the necessity for the solution to beisotonic to blood. A preferred way to therefore maximize ionic strengthfor a given osmolality is to use a poly-ionic species. Interferon-betasolutions of the invention are isotonic to blood (about 290milliosmols/kilogram).

The most preferred stabilizing agent for the present invention is anamino acid that may include one of the following: any acidic amino acid(e.g., glutamic acid, aspartic acid) or an amino acid selected fromarginine and glycine. Most preferably, the amino acid stabilizing agentis arginine which is incorporated as its acidic form (arginine-HCl) inpH 5.0 solutions. A preferred acidic amino acid is L-glutamic acid.Without wishing to be bound by any theory, the fact that poly-ionicexcipients are preferred is probably why arginine and lysine (with 3charged groups) stabilize interferon better than glycine (with 2 chargedgroups), which in turn stabilizes better than any of the unchargedspecies tested.

If the excipient is arginine-HCl, its concentration will range between0.5% (w/v) to 5% and is most preferably 3.13% (equivalent to 150 mMarginine-HCl). If the excipient is glycine, its concentration will rangebetween 0.50% (w/v) to 2.0% and most preferably 0.52% (equivalent to66.7 mM to 266.4 mM, and most preferably 70 mM). If the excipient isglutamic acid, its concentration will range between 100 mM to 200 mM,and is most preferably 170 mM (equivalent to a w/v percent ranging from1.47% to 2.94% and most preferably 2.5%).

We analyzed different excipients as a stabilizing agent for liquidformulations of interferon-beta using the pH buffer system of 50 mMsodium acetate and glacial acetic acid in combination with 100 mM sodiumchloride, pH 5.0. Liquid interferon samples are either thermallystressed by incubation at 37 degrees C. for about 1 to 3 weeks or placedon a rotator for 1 to 3 days as a mechanical stress. Treated samples areevaluated for interferon-beta stability by the methods described inExample 1. As described in more detail in Example 7, the formulationsbuffered at pH 5.0 with sodium acetate containing an amino acidexcipient (and optionally containing sodium chloride) show the beststability.

2. The Interferon

The preferred interferon is fibroblast interferon-beta, most preferablyas recombinant human interferon-beta produced from mammalian cells. Therecombinant human interferon-beta may contain a free sulfhydryl and atleast one disulfide bond. A particularly preferred molecule contains onefree sulfhydryl at position 17 and one disulfide bond between positions31 and 141 per molecule. As is known to be the case with natural humanIFN beta, N-glycosylation is expected at Asn-80. The range ofconcentration in the liquid formulations of the invention is from about30 ug/ml to about 250 ug/ml. A preferred concentration range is 48 to 78ug/ml and the most preferred concentration is about 60 ug/ml. In termsof International Standard values, the Biogen internal standard has beenstandardized to the WHO International Standard for Interferon, Natural#Gb-23-902-531, so that the range of concentration in IU (for a 0.5 mlinjection volume) is from about 6 IMU to 50 IMU and the most preferredconcentration is 12 IMU.

3. The Buffer

The organic acid and phosphate buffers to be used in the presentinvention to maintain the pH in the range of about 4.0 to 7.2 andpreferably from about 4.5 to about 5.5, and most preferably 5.0, can beconventional buffers of organic acids and salts thereof such as citratebuffers (e.g., monosodium citrate-disodium citrate mixture, citricacid-trisodium citrate mixture, citric acid-monosodium citrate mixture,etc.), succinate buffers (e.g., succinic acid-monosodium succinatemixture, succinic acid-sodium hydroxide mixture, succinic acid-disodiumsuccinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodiumtartrate mixture, tartaric acid-potassium tartrate mixture, tartaricacid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaricacid-monosodium fumarate mixture, fumaric acid-disodium fumaratemixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconatebuffers (e.g., gluconic acid-sodium gluconate mixture, gluconicacid-sodium hydroxide mixture, gluconic acid-potassium gluconatemixture, etc.), oxalate buffers (e.g., oxalic acid-sodium oxalatemixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassiumoxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodiumlactate mixture, lactic acid-sodium hydroxide mixture, lacticacid-potassium lactate mixture, etc.) phosphate buffers (sodiumphosphate monobasic/sodium phosphate dibasic) and acetate buffers (e.g.,acetic acid-sodium acetate mixture, acetic acid-sodium hydroxidemixture, etc.).

In the Examples described below, we use different buffer concentrationsand different pHs of sodium phosphate, sodium citrate, sodium succinate,sodium carbonate and sodium acetate for evaluation of the mostappropriate buffer. Interferon-beta samples are either placed at 37degrees C. for 6 days to 2 weeks or placed on a rotator for 7 to 9 hoursin order to accelerate degradative processes. Chemical properties of thesamples are then determined. The samples are analyzed by opticaldensity, peptide mapping, Size Exclusion HPLC, reduced and non-reducedSDS-PAGE/Western blots, and isoelectric focussing/Western blots (IEF),all described below in Example 1. All experimental interferon-betasamples are compared to the starting interferon-beta material or tointerferon-beta samples placed between 2 and 8 degrees C. Our dataindicate that pH is the major factor that determines the stability ofour interferon-beta samples and that samples between pH 4.0 and 5.0 aremore stable than those of pH 7.0 or greater. See Example 2.Nevertheless, we were able to develop several interferon-betaformulations at physiological pH (pH 7.2). See Example 6.

4. Cavitation

Most free sulfhydryl residues in interferon-beta undergo oxidation athigh pH (pH>8.0), the pH at which disulfide bonds undergo rearrangement.We have detected some aggregation of interferon-beta in our bulkintermediate by size-exclusion chromatography, non-reduced SDS-PAGE andlaser light scattering. We have subsequently discovered that formationof aggregated interferon-beta may be dependent upon the level ofdissolved oxygen. The process criteria that we have developed forensuring that the liquid interferon-beta formulations are not cavitatedinclude: (a) if possible, there should be no oxygen-containinggas/liquid interface present during preparation and storage; and/or (b)there should be no bubbles formed during preparation and storage; and/or(c) the levels of dissolved oxygen in the formulation should be keptbelow 10% of atmospheric equilibrium at the preparation and storagetemperature. See Example 3.

5. Adsorption of Interferon to Surfaces

We also determined that interferon will adsorb to certain surfaces andits storage in a glass vessel requires that at least one surface of thevessel in contact with the interferon be coated or otherwise coveredwith a material that will prevent or substantially eliminate theadsorption. This surface may be chemically or physically inert toadsorption. Exemplary materials for this purpose are known to those ofordinary skill in the art and may include, for example, sprayed or bakedsilicone, polypropylene, or polytetrafluoroethylene (PTFE). We took ourpreferred 60 ug/ml liquid formulations (BG9589-1, 2, 3, and 4:summarized in Table 1, below) and filled them into 1 ml long, Type Iglass syringes coated with sprayed silicone (Beckon Dickinson) and into0.75 ml Type I glass vials. The samples are then analyzed by reversephase HPLC (rpHPLC) for protein concentration determination. The dataindicate that there was less protein in solution in those samples thatwere filled into the glass vials as compared to the silicone-coatedprefilled syringes. See Example 5.

We performed kinetic analysis of protein stability using the four liquidformulations whose final concentrations are shown below in Table 1, eachcontaining 60 ug/ml interferon-beta. Alternate formulations, somecontaining surfactants such as PLURONIC® F68 (mfg. by BASF) are given inTable 2.

TABLE 1 Preferred Formulations pH SYSTEM EXCIPIENT FINAL pH 20 mMacetate 150 mM arginine-HCl 5.0 (“BG9589-1”) 20 mM acetate 70 mM glycine5.0 (“BG9589-2”) 100 mM sodium chloride 20 mM phosphate 140 mMarginine-HCl 7.2 (“BG9589-3”) 20 mM phosphate 70 mM glycine 7.2(“BG9589-4”) 100 mM sodium chlorideAll formulation constituents are USP-grade materials. The detailedcompositions are:

BG9589-1 Ingredient (as raw materials) Amount Arginine-HCl, USP 15.8 mgGlacial acetate acid, USP 0.167 mg Sodium acetate trihydrate, USP 0.972mg Interferon-beta 30 ugm Water for Injection, USP 0.5 ml

BG9589-2 Ingredient (as raw materials) Amount Glycine, USP 2.628 mgGlacial acetate acid, USP 0.185 mg Sodium acetate trihydrate, USP 0.932mg Interferonbeta-1a 30 ugm Water for Injection, USP 0.5 ml SodiumChloride 2.922 mg

BG9589-3 Ingredient (as raw materials) Amount Arginine-HCl, USP 14.725mg Sodium phosphate dibasic-7H20 2.332 mg Sodium phosphate monobasic-1H20 0.359 Interferonbeta-1a 30 ug Water for Injection, USP 0.5 ml

BG9589-4 Ingredient (as raw materials) Amount Sodium phosphatedibasic-7H20 1.984 mg Sodium phosphate monobasic-1 H20 0.359 mgInterferonbeta-1a 30 ug Glycine 2.628 mg Sodium Chloride 2.922 mg Waterfor Injection, USP 0.5 ml

TABLE 2 Alternate Formulations pH SYSTEM EXCIPIENT FINAL pH 20 mMacetate 150 mM arginine-HCl and 5.0 15 mg/ml human serum albumin 20 mMacetate 150 mM arginine-HCl and 5.0 0.1% PLURONIC ® F-68 surfactant 20mM acetate 140 mM sodium chloride 5.0 20 mM acetate 15 mg/ml human serum5.0 albumin 140 mM sodium chloride 20 mM acetate 0.1% PLURONIC ® F-685.0 surfactant 140 mM sodium chloride 170 mM L-glutamic acid, 15 mg/mlhuman serum 5.0 150 mM sodium hydroxide albumin 170 mM L-glutamic acid,150 0.1% PLURONIC ® F-68 5.0 mM sodium hydroxide surfactant

Other materials may be incorporated into the formulations of thisinvention. These may include the following preservatives, where allpreferred percentages are w/v: phenol (about 0.2%); methylparaben(0.08%); propylparaben (0.008%); m-cresol (0.1%); chlorobutanol (0.25%);benzyl alcohol (0.1%); and thimerosal (0.1%). Based on analyses todetermine protein aggregation and deamidation (data not presented), themost preferred preservatives are chlorobutanol and benzyl alcohol.

7. Kits for Parenteral Administration

Preferred embodiments of the invention include a packaged kit forparenteral administration of the present liquid formulations. Thepackage may include syringes pre-filled with the liquid formulations ofthe invention, several alcohol swabs, at least one needle, one or moreadhesive bandages and directions for use. It will also appreciated thatthe present liquid formulations of the invention may be used withconventional needleless-injection systems.

E. Using Interferons

The interferon formulations of this invention have antiviral activity.See Example 7. For clinical use, the amount of interferon which isadministered in any particular case, as well as the frequency at whichthe interferon is administered, depends upon such factors as the type ofinterferon used, the disease being treated, and the patient's responseto interferon treatment.

A preferred use of the liquid compositions of the invention is for thetreatment of relapsing multiple sclerosis. Lyophilized (i.e.,reconstituted) liquid formulations of natural interferon-beta andrecombinant interferon-beta have been administered to patients sufferingfrom relapsing multiple sclerosis. See Jacobs et al., Annals ofNeurology 39: 285-294 (March 1996) and references cited therein andJacobs and Munschauer, “Treatment of multiple sclerosis withinterferons” (pp. 223-250) in Treatment of multiple sclerosis: trialdesign, results and future perspectives, (R. A. Rudnick et al., eds),London: Springer, 1992. Use of the liquid formulations described hereinfor treating multiple sclerosis follows the same protocols and measuresthe same primary outcome variables as described in the Jacobs et al.paper, supra.

One way to assess the utility of the present liquid formulations is toperform a toxicology study and assess tissue irritation associated withadministration of the liquid formulation. We have performed a toxicologystudy of the present liquid formulations in rabbits. See Example 8.

The following examples are offered to illustrate embodiments of theinvention, but should not be viewed as limiting the scope of theinvention.

Example 1 Assay Methods

Several well-characterized methods are used to determine thephysico-chemical properties of the interferon-beta in our liquidformulations and these methods may be used to monitor properties ofother interferons as well.

The presence/absence of insoluble aggregate is monitored by measuringthe absorbance at 320 nm and transmittance at 580 nm. The concentrationof soluble protein is determined by either measurement of absorbance at278-280 nm (with an extinction coefficient of 1.5) or by reverse-phasehigh performance liquid chromatography (HPLC) using known concentrationsof interferon-beta spiked in the formulation buffer as standards. Theliquid formulation samples are centrifuged prior to assay. The solubleaggregate percentage is determined by separating aggregates frominterferon-beta monomer by size exclusion chromatography on a TSK-Gel®G2000SWXL column (Toso Haas, Montgomeryville, Pa.). The peak areasmonitored at 280 nm are used to calculated the percentage solubleaggregate.

The stability of the peptide backbone is confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Interferon-betais reduced with mercaptoethanol in the presence of sodium dodecylsulfate before being submitted to electrophoresis on a 10-20% gradientgel (MINIPLUS SEPRAGEL®, Integrated Separation Systems, Natick, Mass.).The proteins are then transferred electrophoretically to anitrocellulose membrane and developed by immunodetection usinganti-interferon-beta antibody and goat anti-mouse antibody coupled tohorseradish peroxidase. See, for example, Gel Electrophoresis ofProteins, A Practical Approach, 2nd edition, B. D. Hames and D.Rickwood, IRL Press.

The change in the net surface charge, caused by deamidation and otherchemical changes, is monitored by isoelectric focusing on apolyacrylamide gel (IEF 3-10 MINIPLUS SEPRAGEL®, Integrated SeparationSystems). See, Gel Electrophoresis of Proteins, A Practical Approach,id.

Methionine oxidation, asparagine deamidation and other possible chemicalchanges are also monitored by peptide mapping. Interferon-beta isdigested with Endoproteinase Lys-C (Wako Pure Chemicals) in the presenceof dithiothreitol and the resulting peptide fragments are separated byreverse-phase HPLC. See generally, Kalgahtgi, K., & Horvath, C. “RapidPeptide Mapping by High Performance Liquid Chromatography”, J.Chromatography 443, 343-354 (1988).

The N-linked oligosaccharide profile is determined by using aFluorophore-Assisted-Carbohydrate-Electrophoresis (FACE®) system byGlyko, Inc. (Novato, Calif.). The asparagine linked (N-linked)oligosaccharides are released from the glycoprotein using the enzymePeptide N-glycosidase F, then labeled with a fluorophore at the reducingtermini by reductive amination, separated and then quantified on apolyacrylamide gel.

Antiviral activity of interferons is determined by a number of methodssuch as those described more fully in: W. E. Stewart II, The InterferonSystem, Springer-Verlag (2d Ed. 1981). The Cytopathic Effect InhibitionAssay (CPE) is particularly useful for determining interferon antiviralactivity. Our preferred method is described in WHO Technical ReportSeries No. 725, Annex 1, (1985), incorporated herein by reference.Briefly, this CPE method is initiated by preparing a working stock ofinterferon-beta standard that has been previously calibrated against aWHO reference standard. This stock is prepared in D-MEM+ mediumcontaining 10% fetal bovine serum and 4 mM L-glutamine at aconcentration of 10,000 units (U) per ml. On the day of assay, standard,control and samples are diluted into D-MEM+ in three separate dilutionseries: a) starting at 32 U/ml followed by 2-fold dilutions; b) startingat 12 U/ml followed by 1.5-fold dilutions; and c) starting at 6 U/mlfollowed by 1.2-fold dilutions. Fifty microliters of the dilutions areadded in columns to the wells of 96-well microliter plates, one plateper dilution series. Next, A549 cells (ATCC Catalog Number CCL-185,Rockville, Md.) in D-MEM+ are added to each well at 5×10⁵ cells/ml, 50micro liters per well, effecting a two-fold dilution of both cells andinterferon-beta. The cells and interferon are incubated at 37 degrees C.in 5% carbon dioxide for 15 to 20 hours. The plate contents are shakeninto a bleach bucket and 100 microliters EMC (encephalomyocarditis)virus at appropriate dilution in media are added to each well. The virusand cells are incubated at 37 degrees C. and 5% carbon dioxide for 30hours. The plate contents are shaken into a bleach bucket, and 0.75%crystal violet dye is added to the plates. After 5 to 10 minutes, theplates are washed with distilled water and allowed to dry. Each assayplate includes cell growth control wells containing neither interferonnor EMC, virus control wells containing EMC and cells but no interferon;and a dilution series of interferon standard.

Plates are examined visually to determine the last well in each columnwith viable cells (>25% confluent purple staining). The detection limitis determined as the lowest concentration of standard which protectsfrom virus cytotoxicity. The sample dilution in the last positive wellis multiplied by the detection limit determined for the standard and thesample dilution factor to obtain the interferon activity (MU/ml) in thesample. Results from each plate are transformed to log units fordetermination of geometric mean and calculation of 95% confidenceintervals.

Example 2 Choice of Buffer System

We prepared three sets of buffers containing between nine and 10different components for each set. Set I contains a series of sodiumphosphate and/or 100 mM sodium chloride solutions between pH 4.0 and7.2. Set II contains an additional series of sodium citrate buffersbetween pH 4.0 and 7.0. Set III contains a series of sodium succinate,sodium acetate and sodium carbonate buffer solutions, all combined with100 mM sodium chloride, having pH values ranging from 4.0 to 7.2. Twoother solutions replaced the sodium chloride with 50 mM sodium sulfateat a pH of 4.0 and 7.2.

Thawed, bulk interferon-beta is dialyzed into different buffersovernight at 2-8 degrees C. with at least two buffer exchanges, thensterile filtered prior to use. Protein concentrations are determined byabsorbance at 278 nm (with extinction coefficient of 1.5 mg⁻¹ ml·cm⁻¹)and all samples contained 140 ug/ml or 150 ug/ml interferon-beta.Samples are filtered and split into four sets by partially filling 2.2ml eppendorf tubes. One set is placed at 2-8 degrees C.; one set wasplaced at 37 degrees C. for 6 days to two weeks; another set is placedon a rotator for 7 to 9 hours; and the final set is used as thezero-time control. Percentage loss of protein due to insolubleaggregates is calculated by the loss of the protein concentration duringvarious treatments divided by the starting concentration.

Results:

The percentage protein loss by insoluble aggregates is calculated asloss of protein concentration divided by the starting proteinconcentration. A statistical analysis of all the data indicates that theinterferon samples in buffers of pH 4.0 and 5.0 had a lower percentageloss of protein due to aggregation than those of higher pH. Interferonsamples incubated at 37 degrees C. and pH 4.0 and 5.0 lost between about10% and 15% due to aggregation. At pH values greater than 6.0, lossesincreased up to 40-50%. We also determined that the interferon sampleshave more soluble aggregates at pH values greater than 6.0. Moreover, wehave determined by peptide mapping that as pH increases from 4.0 to 7.2,there is a substantially linear increase in the amount of interferonthat is deamidated; at pH's 7.0 and higher, greater than 85% ofinterferon is deamidated during the study. We measured the isoelectricpoint (pI) of the protein species in the sample (i.e., that pH at whichthe protein does not migrate in an electric field and the mean charge onthe protein is zero) with IEF/Western blots and the blots show extra pIbands of the samples in sodium citrate and a shifting of band intensityfor samples in sodium succinate. Phosphate has no buffering capacity atpH 5.0. Sodium acetate with sodium chloride at pH 5.0 showed no changein banding pattern or intensity.

Example 3 The Effect of Cavitation

During our pH screening experiments described in Example 2, wediscovered that the head space of the storage tubes appears to becritical for loss of protein of some of the samples. With 1.5 ml of thesamples in 2.2 ml volume tubes, no loss of protein was observed. On thecontrary, 1.2 ml of sample produced significant increase in aggregates.This is consistent with our observations that formation of aggregatedinterferon-beta during the viral inactivation step of the purificationprocess is dependent on the level of dissolved oxygen during this step.

In brief, the viral inactivation step involves adjusting the pH of thechelating SEPHAROSE® agarose bead eluate (see Section C) from7.85+/−0.25 to between 2.5 to 3.5 with 15% phosphoric acid, holding theacidified eluate for 120-135 minutes, and then readjusting the pH to6.7+/−0.7 with 0.5 N sodium hydroxide. All steps are performed at 2-8degrees C. We designed a study to determine if a relationship existsbetween formation of interferon-beta aggregates in this step and theamount of dissolved oxygen.

Material and Methods

Eluate from the chelating SEPHAROSE® AGAROSE BEAD column is divided into50 ml or 100 ml aliquots and placed into 100 ml spinner flasks. To eachflask, 1 ml of argon-sparged 15% phosphoric acid is added. The flask isthen gently stirred for about 2 minutes, and held without stirring forabout 2 hours at 2-8 degrees C. Following this hold period, 6.5 ml ofargon-sparged sodium hydroxide is added and the sample assayed bysize-exclusion chromatography at various times. Dissolved oxygen withinthe liquid is measured continuously with an oxygen probe (Orion, Model860) and recorded at the time of base addition. For samples withdissolved oxygen levels equal to or less than 10%, argon gas is sweptthrough the reaction vessel head space.

Results: Data are presented in FIG. 1 which reveal a clear relationshipbetween the amount of dissolved oxygen present at the time of sodiumhydroxide addition and the yield of interferon-beta monomer through thevirus inactivation step. The yield values obtained at dissolved oxygenconcentrations less than or equal to 10% are significantly differentfrom all other yields at other oxygen concentrations. We alsocharacterized the aggregate (data not presented here) and determinedthat its specific activity is reduced about 30-40 fold from the bulkintermediate. We also determined that greater than about 90% of theaggregate is resistant to SDS denaturation under non-reducingconditions, suggesting a covalent cross-linkage. Under reducingconditions (2% beta-mercaptoethanol) the aggregate collapses to themonomer, suggesting cross-linkage that involves disulfide bonds.

Example 4 Choice of Excipient

A series of interferon-beta (60 ug/ml) formulations containing differentexcipients are prepared in a preferred pH 5.0 buffer containing 50 mMsodium acetate and 100 mM sodium chloride. The excipients includeglycine, arginine-HCl, lysine-HCl, sucrose, glycerin, PEG3350,glutathione and PLURONIC® F68 surfactant. Interferon-beta bulkintermediate is dialyzed into 50 mM sodium acetate and 100 mM sodiumchloride, pH 5.0 overnight at 2-8 degrees C. with at least two bufferexchanges, then filtered prior to use. Interferon-beta concentrationsare determined by absorbance at 278 nm with background subtraction. Allsamples are diluted to final interferon concentrations of about 60ug/ml. All prepared samples are filtered, two milliliters aretransferred to 4 ml glass vials (non-siliconed), the head space spargedwith argon and the vials sealed. Sets of samples are placed at 2-8degrees C. and 37 degrees C. for periods up to two weeks. Other samplesare mechanically stressed by rotating them at room temperature for 3days.

Samples are analyzed according to the procedures of Example 1. Inaddition, the percentage of dissolved oxygen in the formulations ismeasured by a Ciba-Corning Model 248 blood gas analyzer. The“experimental” value is the oxygen partial pressure (mm Hg) of thesamples minus that of the nitrogen purged buffer blank and the “control”value is the partial pressure of oxygen in the buffer blank stored atroom temperature minus the oxygen partial pressure of the nitrogenpurged buffer blank. The percentage dissolved oxygen(“experimental”/“control”) is always less than 30%.

Results:

IEF/Western blots and SDS-PAGE/Western blots of samples incubated at 37degrees C. for two weeks indicate band shifting and loss of intensity aswell as the presence of interferon multimers in samples containingPEG3350 and glutathione. After an additional week at 37 degrees C.,glycerin excipient shows one extra band in our blots. Sucrose excipientshows loss of band intensity. This initial screening procedure allowedus to consider in more detail arginine-HCl, glycine, sodium chloride andmannitol for further studies.

Example 5 Adsorption of Interferon

Thawed bulk interferon-beta is dialyzed to BG9589-1, 2, 3 and 4 (seeTable 1) overnight at 2-8° C. with at least two buffer exchanges, thenfiltered prior to use. The protein concentrations are determined byabsorbance at 280 nm (with extinction coefficient of 1.5 mg⁻¹ml·cm⁻¹).All the samples are diluted to final concentrations of approximately of60 ug/ml. The diluted samples are filtered and filled either 0.5 ml intotriplicate, 1.0 ml long, sprayed silicon BD syringes (Type I glass) withnitrogen flushed headspace or 0.75 ml into triplicate, 0.75 ml Type Iglass vials with argon flushed headspace. Protein concentrations aredetermined by reverse phase HPLC (Example 1).

Results:

Table 3 below lists the protein concentrations that were determined byreverse phase HPLC. The data indicate that there is less protein for thesamples that were filled into the glass vials as compared to the siliconcoated prefilled syringes. Thus, siliconized syringes are used for theliquid formulation of interferon-beta.

TABLE 3 Glass vial Siliconized Syringes (ug/ml) (S.D) (ug/ml) (S.D)BG9589-1 59.3 (2.6) 63.3 (2.5) BG9589-2 58.3 (0.7) 61.7 (0.1) BG9589-356.4 (0.4) 58.8 (1.1) BG9589-4 55.5 (0.7) 59.3 (0.5)

Example 6 Formulations at Physiological pH

Ionic Strength/Phosphate. We carried out initial studies inphosphate/sodium chloride, pH 7.2 buffer systems of varying buffercomponent concentrations in which the phosphate concentration variedbetween 10, 50 and 75 mM with an ionic strength (defined by I=Σc₁z₁ ²,where c₁ and z₁ are the molar concentration and valence charge of ionicspecies I, respectively) of 0.2, 0.4 and 0.6, adjusted by addition ofsodium chloride.

We used a full factorial design on the variables of phosphateconcentration (10, 50 and 75 mM) and ionic strength (I=0.2, 0.4, and0.6). Compositions of sodium phosphate monobasic, sodium phosphatedibasic and sodium chloride (to achieve the desired ionic strength) inthe buffers are calculated using a spreadsheet adapted from Ellis andMorrison, “Buffers of Constant Ionic Strength for Studying pH-dependentProcesses”, Methods Enzymol. 87: 405-426 (1982). The equations alloweddetermination of requisite amounts of each buffer component forspecified pH, phosphate concentration and ionic strength. Each of thenine solutions used in the factorial experiment is obtained by bufferexchange of interferon-beta bulk intermediate through Pharmacia PD-10desalting columns. The pHs of all resulting solutions are at7.20+/−0.15. Concentrations are assayed by absorbance at 280 nm and thendiluted to 150 ug/ml interferon-beta with the appropriate buffer. Theresulting solutions are sterile filtered under argon through 0.22 micronfilters, and 1.3 ml is aliquoted into 5 ml glass vials with an argonhead space. Samples are incubated at 37 degrees C. for 6 days and run intriplicate. Samples are analyzed by percent transmittance at 580 nm,percent protein recovery, and IEF-PAGE/Western blots.

Results:

Analysis of percentage transmittance with respect to varying ionicstrength shows a trend toward increasing transmittance (i.e, decreasingamounts of insoluble protein aggregates) with increasing ionic strength.Percent protein recovery data shows a similar trend although IEF-PAGEWestern blots show no trend in deamidation with varying ionic strengthso that all the samples are equally deamidated. Thus, after storage forsix days at 37 C, samples tended to show less aggregation withdecreasing phosphate concentration and increasing ionic strength. Theresults of the experiments on the percentage transmittance and percentrecovery as a function of varying phosphate concentration (not presentedhere) show a weak trend towards decreasing % transmittance withincreasing phosphate concentration, but an analysis of variance shows nosignificant difference in the means of samples with different phosphateconcentrations. The percentage recovery data show improved proteinrecovery for lower phosphate concentrations (a significant difference atthe 94% confidence level). IEF-PAGE Western blots display no discernibletrend in deamidation with varying phosphate concentration.

Excipient/Salt Ratio. Preliminary studies (not shown) indicated thatsome excipients may require salts (e.g., sodium chloride) in order tomaintain high ionic strength and in order to exhibit a stabilizingeffect at pH 7.2. We designed a factorial study using excipients(glycine, lysine, arginine, sucrose and mannitol) and fraction of sodiumchloride contributing to isotonicity (f_(salt)=0, 0.25, 0.75 and 1.0).The fraction is calculated by:f_(salt)=O_(salt)/(O_(salt)+O_(excipient)), where O_(salt) andO_(excipient) are the osmolalities in mOsm/kg of the sodium chloride andexcipient, respectively, in the solution. Salt fraction provides a meansof comparing salt effects across different excipients. All samplescontained additives to isotonicity, with varying ratios ofexcipient:salt (as defined by f_(salt)).

Ten percent (w/v) stock solutions of each excipient in 20 mM phosphate,pH 7.2, are prepared, degassed, and sparged with argon. A stock solutionof 250 mM sodium chloride, 20 mM phosphate, pH 7.2 is prepared, degassedand sparged with argon. Bulk interferon-beta intermediate is extensivelydialyzed against argon-sparged 20 mM phosphate, pH 7.2 buffer. Theresultant solution is assayed for interferon-beta concentration byabsorbance at 280 nm and diluted with phosphate buffer and respectivestock solutions of excipient and salt to achieve 60 ug/mlinterferon-beta and the desired final salt and excipient conditions. Theresulting samples are filter sterilized (0.22 micron) and filled into1.0 ml Becton Dickinson sprayed silicone, Type I glass syringes (0.5 mlfill volume) with a nitrogen head space. Samples are stored at 40degrees C.

At 6 days, arginine, glycine and sucrose samples are analyzed byabsorbance at 320 and 280 nm, both before and after filtration through0.22 micron filters. At 2 weeks, arginine, lysine and mannitol aresimilarly analyzed, along with IEF-PAGE, reducing SDS-PAGE andnon-reducing SDS-PAGE. Control samples were stored at between 2 and 8degrees C. and analyzed similarly.

Results:

The recovery of Interferon-beta 1a (as percentage of control) increaseswith increasing f salt for sucrose and mannitol, reaching a maximumrecovery at f_(salt)=1 (130 mM sodium chloride). For arginine andlysine, recovery decreases with increasing f_(salt). Maximum recoveryfor glycine formulations at pH 7.2 is reached at about f_(salt)=0.75.

This excipient screening study using a pH 7.2 phosphate buffer withvarious excipients such as glycine, lysine, arginine, mannitol andsucrose added to isotonicity, showed poor recovery for all non-chargedexcipients. The extent of deamidation was not affected by theseadditives. For instance, reducing and non-reducing SDS/PAGE indicateloss of non-glycosylated interferon-beta species in all formulations,and heavier multimer bands for isotonic sodium chloride alone andmannitol. In sum, there is a thus a strong correlation between the ioniccharacter of the excipient and its ability to stabilize interferon-betaagainst aggregation in these buffer systems at physiological pH.Non-ionic additives such as sucrose and mannitol appear to offer noprotection, or may actually promote protein loss at physiological pH.Sodium chloride, with a single charge per soluble species, performsbetter than either of mannitol or sucrose. Amino acids contain twocharges per molecule at physiological pH. In the case of glycine, thezwitterionic nature of the molecule itself does not seem to besufficient enough to stabilize interferon-beta. Arginine and lysine,each containing three charges per molecule, stabilize interferon-betabetter than either sodium chloride alone or glycine/sodium chlorideformulations.

Example 7 Stability and Kinetic Studies

Formulations are aseptically filled under an inert atmosphere, syringesincubated at a range of temperatures for varying time periods andsyringe contents are analyzed. In brief, thawed bulk interferon-beta isdialyzed to BG9589-1, -2, -3 and -4 overnight at 2-8 degrees C. with aleast two buffer exchanges. Protein concentrations are determined byabsorbance at 280 nm with an extinction coefficient of 1.5 ml/mg/cm. Allsamples are diluted to a final Interferon-beta-1a concentration of about60 ug/ml. The four Interferon-beta-1a formulations of Table 1 arefiltered and 0.5 ml are dispensed into 1.0 ml long, Becton Dickinson(BD) syringes whose interior surfaces were coated with baked silicone orwith sprayed silicone. The samples were analyzed by OD, size exclusionHPLC (SEC), Isoelectric focusing gel electrophoresis (IEF)/western blot,reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)/western blot, peptide mapping, fluorophore assistedcarbohydrate electrophoresis (FACE®) and CPE bioassay. The head space inthe syringe is nitrogen gas. The syringes are incubated at 2-8 degreesC., 25 degrees C., 33 degrees C. and 40 degrees C. for up to ninetydays. Samples are analyzed according to the methods in Example 1.

Results:

We analyzed protein concentrations of our samples, normalized againstthat of the starting material for periods up to ninety days at a varietyof temperatures. FIG. 2 illustrates that BG9589-1 showed completeprotein stability (no loss of protein) after 3 months incubation attemperatures ranging from 2-8 degrees C. (average 4 degrees C.) up to 25degrees C. At a storage temperature (33 degrees C.) approaching bodytemperature, about 18% of the protein was degraded. At a storagetemperature (40 degrees C.) exceeding body temperature, about thirtypercent of protein was degraded at the end of 3 months. Substantiallyidentical results were obtained for BG9589-2 (not shown). FIG. 3illustrates results of 2 month storage tests on BG9589-3. Proteindegradation was minimal at 4 through 25 degrees C. but was rapid athigher temperatures. Results for BG9589-4 are substantially identical tothose in FIGS. 2 and 3. These data were confirmed using reducedSDS-PAGE/Western blots.

In the “baked” syringes, over the period of this study there are nodetectable soluble aggregates. No significant changes are observed inprotein concentration, CPE assay, percent Oxidized AP6 and carbohydrateprofiles. There are no observable changes in the samples as seen byreducing SDS-PAGE/western blot and IEF/western blot. There is someincrease in percent deamidation as compared to the starting time point.However, the bulk intermediate that was used to fill these syringes has37% deamidation, which is higher than the 33.8% value of the materialafter being filled into the syringes. This latter, low value may be dueto assay variability. In the “sprayed” syringes over the period of thisstudy, there are also no detectable soluble aggregates. No significantchanges are observed in protein concentration, CPE assay, percentdeamidation, percent Oxidized AP6 and carbohydrate profiles. There areno observable changes in the samples as seen by reducingSDS-P-AGE/western blot and IEF/western blot. In short, results so farhave shown that final product BG9589-1 is stable up to 3 months at 2-8degrees C. in the “baked silicone” syringes, and 6 months at 2-8 degreesC. in “sprayed silicone” syringes.

We performed the antiviral CPE assay on formulations BG9589-1 andBG9589-2 (see Table 1) after syringes were aseptically filled. Reportedactivity values for both BG9589-1 and BG9589-2 are 12.0 MU/ml. Theantiviral CPE assay was repeated after storage of the samples for up to3 months at between 2-8 degrees C. The reported activity values forBG9589-1 are 11.6 MU/ml (n=8) with a 95% confidence interval of10.2-13.3 MU/ml.

We also measured stability of the bulk intermediate material of BG9589-1at 2-8 degrees C. for 5 months, and −70 degrees C. for 6 months. Samplesof BG9589-1 from pilot diafiltration studies were analyzed by themethods of Example 1. Results so far have shown that in-process materialof BG9589-1 is stable at 2-8 degrees C. for 5 months, and at −70 degreesC. for 6 months.

Over the period of this particular study, there are no detectablesoluble aggregates. No significant changes are observed for percentdeamidation and carbohydrate profiles (The differences in percentdeamidation are within assay variability). There are no observablechanges in the samples as seen by reducing SDS-PAGE/western blot andIEF/western blot. There is a slight decrease in the proteinconcentration. The decrease in protein concentration for the −70 degreesC. may due to the sample going through one freeze/thaw cycle. Thedecrease of protein concentration is still within 15% of the initialconcentration.

Example 8 Preclinical Studies

A single intramuscular (IM) dose local tolerability study in rabbits isconducted which evaluates the local toxicity of interferon whenadministered in several new formulations. Injection site reactions dueto administration of the present liquid formulation or with lyophilizedand reconstituted interferon formulations are comparable to thoseevident following administration of normal saline.

1. Rabbit Irritation/Bioavailability Study Following Single Dose IMAdministration of Four Formulations of Interferon-Beta)

Twenty male New Zealand white rabbits each received a single 30 ugintramuscular (IM) injection of Interferon beta-1a as one of fiveformulations: BG9589-1 (pH 5.0, acetate buffer, arginine stabilizer, 0.5ml/dose); BG9589-2 (pH 5.0, acetate buffer, glycine/NaCl stabilizer, 0.5nil/dose); BG9589-3 (pH 7.2, phosphate buffer, arginine stabilizer, 0.5ml/dose); BG9589-4 (pH 7.2, phosphate buffer, glycine/NaCl stabilizer,0.5 ml/dose); and a lyophilized interferon-beta formulation at pH 7.2containing 1.5% HSA, 1.0 ml/dose (See Jacobs et al., supra).

Four animals received each treatment. Animals that received BG9589-1 orthe lyophilized formulation also received an equivalent volume injectionof normal saline in a contralateral site as a negative control. Bloodsamples are collected through 72 hours post-dose for serum interferonbeta activity analyses. Macroscopic dermal evaluations for erythema,scar formation and edema are conducted at 6, 12, 24, 48 and 72 hourspost-dose. Following the 72 hour post-dose blood collection, the animalsare sacrificed, the injection sites are inspected macroscopically forsigns of tissue damage and then fixed in 10% neutral buffered formalin.The muscle samples (three/injection site) are examined microscopicallyfor inflammation, necrosis, hemorrhage and lesions.

Results:

When graded by Primary Irritation Index scores (EPA DermalClassification System), none of the above liquid formulations weredetermined to be more than a slight skin irritant. Macroscopicinspection of a BG9589-4 injection site in one animal indicated slightirritation (hemorrhage); however microscopic investigation revealed nosigns of hemorrhage and the macroscopic observation was determined to bean artifact. In short, microscopic examinations reveals that the liquidformulation test article injection site reactions were consistentlyminimal to mild and that no reaction was more severe than those inducedby administration of the lyophilized formulation or normal saline.

In addition, rabbit dermal irritation following repeated IMadministrations of the liquid formulations may easily be tested usingmultiple groups of rabbits that will receive intramuscular injections ofliquid formulations or normal saline every other day for eight days(five doses total). Doses are administered in a pre-defined area on eachanimal's back to maximize local exposure to test article. Macroscopicdermal evaluations are conducted at 4-6 hours following eachadministration and 24 hours following the last administration for eachtreatment group. Daily gross observations are made at the time of eachdermal evaluation. Following the 24 hour post-dose macroscopicexamination, the animals are sacrificed, the injection sites will beinspected for signs of tissue damage and the tissue fixed in 10% neutralbuffered formalin. The preserved tissues are examined microscopicallyfor inflammation, necrosis, hemorrhage, and lesions. Blood samples alsoare collected immediately prior to the initial test articleadministration and at the time of sacrifice for hematology and serumchemistry evaluations.

Example 9 Clinical Studies

The present liquid formulations differ significantly from priorinterferon formulations. For any clinical indication, there is thepotential for a change in pharmacokinetic and pharmacodynamic behaviorof the interferon when administered to humans. Unfortunately, theactivities of interferon-beta are highly species specific and the mostpertinent pharmacologic information is derived from studies in humancells in culture, in humans, and, to a lesser extent, in rhesus monkeys.A preferred way to test for pharmacological change, if any, is toconduct a human bioequivalence trial.

Anti-viral levels of interferon-beta in serum can be quantitated using acytopathic effect (CPE) bioassay, as described for instance inExample 1. A human bioequivalence study can be conducted with any numberof liquid and lyophilized interferon formulations. Through analysis ofserum, area under the curve (AUC) and C_(MAX) activity parameters, oneof ordinary skill can determine whether lyophilized and liquidformulations are bioequivalent. As but one example of a bioequivalencestudy protocol, we describe briefly a double-blind, single-dose,crossover study to demonstrate the bioequivalence of a liquidformulation of the invention and a lyophilized interferon-beta productin healthy volunteers. Design. Each subject receives the same dosage(e.g., 60 ug/12 MU) of interferon-beta formulations in a double-blind,two-period crossover (Table 4). Subjects are between the ages of 18 and45 years, inclusive and within 15% of the normal body weight range forheight and body frame. Blood samples for hematology, chemistry, seruminterferon beta activity and pharmacodynamic profiles are drawnimmediately prior to, and at various times following, each dose, through144 hours post-dose. Assessment of injection pain and injection sitereactions also is followed.

Study Conduct.

As prophylaxis against interferon-associated flu-syndrome, all subjectswill receive acetaminophen immediately before and throughout the dosingperiods.

Pharmacokinetics.

Serum Interferon Beta Determinations.

Serum levels are measured as units of antiviral activity by a (CPE)assay. Serum antiviral levels are analyzed for AUC, C_(max) and T_(max).AUC values will be calculated from time of dosing to the last detectablelevel (AUC_(0-T)) and through 144 hours post dose (AUC₀₋₄₄₄). Standarddescriptive analysis of the treatment data are conducted using SAS(version 6.08, SAS Institute, Cary, N.C.).

TABLE 5 Dose schedule for Exemplary Study Dose Dose Treatment TreatmentGroup Route (MU) Period: 1 Period: 2 1 IM 12 Lyophilized Liquid (60 mcg)(60 mcg) 2 IM 12 Liquid Lyophilized (60 mcg) (60 mcg)Pharmacodynamics.

The biological marker neopterin, a product of the interferon inducedenzyme GTP cyclohydrolase which reflects macrophage and T-cellactivation (C. Huber et al., J Exp Med 1984; 160: 310-314; Sep. 20,1996; D. Fuchs et al., Immunol. Today 9: 150-155, 1988) has beencharacterized. In both nonclinical and clinical studies of recombinanthuman interferon beta, induction of neopterin correlates with serumactivity levels following administration of various recombinant humaninterferon beta treatments.

Neopterin is measured via standard laboratory procedures. Thepharmacodynamic profile of interferon-beta is described in aquantitative manner by calculation of three serum neopterin parameters.The first parameter, E_(AUC), is the area under the neopterin vs timecurve normalized to baseline level. The second parameter is E_(MAX);this parameter is the difference between the observed peak neopterinlevel and the baseline neopterin level. The third parameter is theinduction ratio, IR; this parameter is calculated as the peak neopterinlevel divided by the baseline neopterin level.

Statistics.

The Wilcoxon-Mann-Whitney two, one-sided tests procedure is used on AUCto determine equivalence. To estimate the relative bioavailability ofinterferon from the liquid formulation relative to the lyophilizedformulation and its 90% confidence limits, AUC is submitted to ananalysis of variance (ANOVA) after logarithmic transformation. From the“between-subject” variation, the sequences and genders are isolated.From the “within-subjects” variation, components due to periods andtreatments are isolated.

EQUIVALENTS

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed therein. It is intended that thespecification and examples be considered exemplary only, with the truescope and spirit of the invention being indicated by the followingclaims.

The invention claimed is:
 1. A liquid formulation comprising: (i) aninterferon-beta, and (ii) a stabilizing agent at between about 0.3% and5% by weight, wherein said stabilizing agent is L-glutamic acid, whereinthe formulation does not comprise serum albumin, and wherein theformulation has not been previously lyophilized.
 2. The formulation ofclaim 1, wherein the formulation has a pH of between about 4.0 and 7.2.3. The formulation of claim 1, wherein the formulation has a pH ofbetween about 4.5 and 5.5.
 4. The formulation of claim 1, wherein theformulation comprises between 100 mM and 200 mM L-glutamic acid.
 5. Theformulation of claim 1, wherein the formulation comprises 170 mML-glutamic acid.
 6. The formulation of claim 1, wherein theinterferon-beta is present at between 6 MIU/ml and 50 MIU/ml.
 7. Theformulation of claim 1, wherein the formulation further comprises sodiumhydroxide.
 8. The formulation of claim 7, wherein the sodium hydroxideis present at 150 mM.
 9. The formulation of claim 1, further comprisinga surfactant.
 10. The formulation of claim 1, wherein said liquidformulation has a dissolved oxygen level that is less than 30% ofatmospheric equilibrium levels.
 11. The formulation of claim 10, whereinsaid liquid formulation has a dissolved oxygen level that is less thanor equal to 10% of atmospheric equilibrium levels.
 12. A packaged kitfor parenteral administration of an interferon-beta, the kit containinga syringe pre-filled with a liquid formulation comprising theinterferon-beta and between about 0.3% and 5% by weight L-glutamic acid;wherein the liquid formulation has not been reconstituted fromlyophilized interferon; wherein the liquid formulation is not furtherlyophilized, wherein the liquid formulation does not comprise serumalbumin, and wherein the syringe has a head space flushed with an inertgas.
 13. The packaged kit of claim 12, wherein at least one surface ofthe syringe in contact with the liquid formulation is coated with amaterial inert to interferon.
 14. The packaged kit of claim 12, whereinthe formulation has not been cavitated.
 15. The packaged kit of claim12, wherein the formulation has a pH of between about 4.0 and 7.2. 16.The packaged kit of claim 12, wherein the formulation has a pH ofbetween about 4.5 and 5.5.
 17. The packaged kit of claim 12, wherein theformulation comprises between 100 mM and 200 mM L-glutamic acid.
 18. Thepackaged kit of claim 12, wherein the formulation comprises 170 mML-glutamic acid.
 19. The packaged kit of claim 12, wherein theinterferon-beta is present at between 6 MIU/ml and 50 MIU/ml.
 20. Thepackaged kit of claim 12, wherein the formulation further comprisessodium hydroxide.
 21. The packaged kit of claim 20, wherein the sodiumhydroxide is present at 150 mM.
 22. The packaged kit of claim 12,wherein the formulation further comprises a surfactant.
 23. The packagedkit of claim 12, wherein said liquid formulation has a dissolved oxygenlevel that is less than 30% of atmospheric equilibrium levels.
 24. Thepackaged kit of claim 23, wherein said liquid formulation has adissolved oxygen level that is less than or equal to 10% of atmosphericequilibrium levels.